Immunogenic composition comprising an il-13 element and t cell epitopes, and its therapeutic use

ABSTRACT

The present invention relates to isolated immunogens and their use in the treatment of diseases that are treatable with neutralization of IL-13, such as COPD, asthma and atopic disorders such as hayfever, contact allergies and atopic dermatitis. In particular the invention relates to the neutralization of the biological effects of IL-13 by raising an immune response against the IL-13 by vaccination of a mammal with immunogens comprising the native or mutated amino acid sequence of IL-13, and foreign T-helper epitopes either inserted in, or attached to the IL-13 sequence or present in carrier polypeptides. Also provided by the present invention are DNA vaccines that comprise a polynucleotide sequence that encodes the immunogens of the present invention. The invention further relates to pharmaceutical compositions comprising such immunogens and their use in medicine and to methods for their production.

The present invention relates to isolated immunogens and their use inthe treatment of diseases that are treatable with neutralisation ofIL-13, such as COPD, asthma and atopic disorders such as hayfever,contact allergies and atopic dermatitis. Most preferably the immunogensare used in the treatment of asthma. In particular the invention relatesto the neutralisation of the biological effects of IL-13 by raising animmune response against the IL-13 by vaccination of a mammal withimmunogens comprising the native or mutated amino acid sequence ofIL-13, and foreign T-helper epitopes either inserted in, or attached tothe IL-13 sequence or present in carrier polypeptides. Also provided bythe present invention are DNA vaccines that comprise a polynucleotidesequence that encodes the immunogens of the present invention. Theinvention further relates to pharmaceutical compositions comprising suchimmunogens and their use in medicine and to methods for theirproduction.

BACKGROUND TO THE INVENTION

COPD is an umbrella term to describe diseases of the respiratory tract,which shows similar symptoms to asthma and is treated with the samedrugs. COPD is characterised by a chronic, progressive and largelyirreversible airflow obstruction. The contribution of the individual tothe course of the disease is unknown, but smoking cigarettes is thoughtto cause 90% of the cases. Symptoms include coughing, chronicbronchitis, breathlessness and respiratory injections. Ultimately thedisease will lead to severe disability and death.

Asthma is a chronic lung disease, caused by inflammation of the lowerairways and is characterised by recurrent breathing problems. Airways ofpatients are sensitive and swollen or inflamed to some degree all thetime, even when there are no symptoms. Inflammation results in narrowingof the airways and reduces the flow of air in and out of the lungs,making breathing difficult and leading to wheezing, chest tightness andcoughing. Asthma is triggered by super-sensitivity towards allergens(e.g. dust mites, pollens, moulds), irritants (e.g. smoke, fumes, strongodours), respiratory infections, exercise and dry weather. The triggersirritate the airways and the lining of the airways swell to become evenmore inflamed, mucus then clogs up the airways and the muscles aroundthe airways tighten up until breathing becomes difficult and stressfuland asthma symptoms appear.

Atopic disorders refers to a group of diseases that are hereditary andoften occur together, including asthma, allergies such as hay fever, andatopic dermatitis. Atopic dermatitis is a chronic disease that affectsthe skin. In atopic dermatitis, the skin becomes extremely itchy andinflamed, causing redness, swelling, cracking, weeping, crusting, andscaling. Atopic dermatitis most often affects infants and youngchildren, but it can continue into adulthood or first show up later inlife. In most cases, there are periods of time when the disease isworse, called exacerbations or flares, followed by periods when the skinimproves or clears up entirely, called remissions. Many children withatopic dermatitis will experience a permanent remission of the diseasewhen they get older, although their skin often remains dry and easilyirritated. Environmental factors can bring on symptoms of atopicdermatitis at any time in the lives of individuals who have inheritedthe atopic disease trait. Atopic dermatitis is often referred to as“eczema,” which is a general term for the many types of dermatitis.Atopic dermatitis is the most common of the many types of eczema.Several have very similar symptoms.

The way the skin is affected by atopic dermatitis can be changed bypatterns of scratching and resulting skin infections. Some people withthe disease develop red, scaling skin where the immune system in theskin is becoming very activated. Others develop thick and leathery skinas a result of constant scratching and rubbing. This condition is calledlichenification. Still others develop papules, or small raised bumps, ontheir skin. When the papules are scratched, they may open (excoriations)and become crusty and infected.

Many factors or conditions can make symptoms of atopic dermatitis worse,further triggering the already overactive immune system in the skin,aggravating the itch-scratch cycle, and increasing damage to the skin.These exacerbating factors can be broken down into two main categories:irritants (such as wool or synthetic fibers, rough or poorly fittingclothing, soaps and detergents, some perfumes and cosmetics, chlorine,mineral oil, some solvents, dust or sand) and allergens (such as pollen,dog or cat dander, and dust mite allergens). Emotional factors and someinfections can also influence atopic dermatitis.

If a flare of atopic dermatitis does occur, several methods can be usedto treat the symptoms. Corticosteroids as topical creams are the mostfrequently used treatment, although systemic administration is also usedin some severe cases. Sometimes over-the-counter preparations are used,but in many cases the doctor will prescribe a stronger corticosteroidcream or ointment. An example of a commonly prescribed corticosteroid isprednisone. Side effects of repeated or long-term use of topicalcorticosteroids can include thinning of the skin, infections, growthsuppression (in children), and stretch marks on the skin. Antibiotics totreat skin infections may be applied directly to the skin in anointment, but are usually more effective when taken by mouth.Phototherapy (treatment with light) that uses ultraviolet A or B lightwaves, or both together, can be an effective treatment for mild tomoderate dermatitis in older children (over 12 years old) and adults. Inadults, immunosuppressive drugs, such as cyclosporine, are also used totreat severe cases of atopic dermatitis that have failed to respond toany other forms of therapy. The side effects of cyclosporine can includehigh blood pressure, nausea, vomiting, kidney problems, headaches,tingling or numbness, and a possible increased risk of cancer andinfections.

Because of the unmet medical need therefor and the side affects ofexisting therapies there is a need for alternative treatments for atopicdiseases in general, and in particular for treatments for asthma andatopic dermatitis.

IL-13 is a Th2-type cytokine that is closely related to IL-4. A numberof recent papers have defined the role for IL-13 in driving pathology inthe ovalbumin model of allergenic asthma (Wills-Karp et al, 1998,Science:282:2258-2261; Grunig et al, 1998, Science 282:2261-2263). Inthis work, mice previously sensitised to ovalbumin were injected with asoluble IL-13 receptor which binds and neutralises IL-13. Airwayhyper-responsiveness to acetylcholine challenge was reduced in thetreated group. Histological analysis revealed that treated mice hadreversed the goblet-cell metaplasia seen in controls. In complementaryexperiments, lung IL-13 levels were raised by over-expression in atransgenic mouse or by installation of protein into the trachea inwild-type mice. In both settings, airway hyper-responsiveness,eosinophil invasion and increased mucus production were seen (Zhu et al,1999, J. Clin. Invest. 103:779-788).

The sequence of the mature form of human IL-13 is provided in SEQ ID No.1 and is shown in FIG. 1.

The sequence of the mature form of murine IL-13 is provided in SEQ IDNo. 2 and is shown in FIG. 2.

Sequences for IL-13 from several mammalian species and non-humanprimates are shown in FIG. 3 and FIG. 4 (SEQ ID NO.s 3 to 9)

As a result of the various problems associated with the production,administration and tolerance of monoclonal antibodies there is anincreased focus on methods of instructing the patient's own immunesystem to generate endogenous antibodies of the appropriate specificityby means of vaccination. However, mammals do not generally havehigh-titre antibodies against self-proteins present in serum, as theimmune system contains homeostatic mechanisms to prevent theirformation. The importance of these “tolerance” mechanisms is illustratedby diseases like myasthenia gravis, in which auto-antibodies directed tothe nicotinic acetylcholine receptor of skeletal muscle cause weaknessand fatigue (Drachman, 1994, N Engl J Med 330:1797-1810).

A number of techniques have been designed with the aim of breaking“tolerance” to self antigen. One technique involves chemicallycross-linking the self-protein (or peptides derived from it) to a highlyimmunogenic carrier protein, such as keyhole limpet haemocyanin(“Antibodies: A laboratory manual” Harlow, E and Lane D. 1988. ColdSpring Harbor Press).

A variant on the carrier protein technique involves the construction ofa gene encoding a fusion protein comprising both carrier protein (forexample hepatitis B core protein) and self-protein (The core antigen ofhepatitis B virus as a carrier for immunogenic peptides”, BiologicalChemistry. 380(3):277-83, 1999). The fusion gene may be administereddirectly as part of a nucleic acid vaccine. Alternatively, it may beexpressed in a suitable host cell in vitro, the gene product purifiedand then delivered as a conventional vaccine, with or without anadjuvant.

Another approach has been described by Dalum and colleagues wherein asingle class II MHC-restricted epitope is inserted into the targetmolecule. They demonstrated the use of this method to induce antibodiesto ubiquitin (Dalum et al, 1996, J Immunol 157:4796-4804; Dalum et al,1997, Mol Immunol 34:1113-1120) and the cytokine TNF (Dalum et al, 1999,Nature Biotech 17:666-669). As a result, all T cell help must ariseeither from this single epitope or from junctional sequences. Such anapproach is also described in EP 0 752 886 B1, WO 95/05849, and WO00/65058.

Treatment therapies, some including vaccination, for the neutralisationof several cytokines are known. WO 00/65058 describes a method of downregulating the function of the cytokine IL-5, and its use in thetreatment of asthma. In this study, the IL-5 sequence was modified by anumber of techniques to render it immunogenic, amongst which there isdescribed an IL-5 immunogen supplemented with foreign T-cell epitopes,whilst maintaining the IL-5 B cell epitopes. WO 01/62287 disclosesIL-13, amongst a long list of potential antigens, for use in allergy orasthma vaccines. WO 00/06937 discloses cytokine derivatives that arefunctionally inactivated for use as vaccine antigens. Chimaeric IL-13immunogens are disclosed in the co-pending patent application WO02/070711.

There remains a need to provide improved immunotherapeutic treatmentsfor asthma, and improved immunogens for raising neutralising anti-IL-13immune responses.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprisingmodified “self” IL-13 immunogens, wherein the IL-13 immunogen ismodified to include foreign T-cell helper epitopes. The pharmaceuticalcomposition is preferably for use in human therapy, and in thiscomposition the IL-13 sequence is a human sequence or other sequencethat is capable of generating an immune response that recognises humanIL-13; and the T-cell helper epitopes are “foreign” with respect tohuman self-proteins. Preferably the T-helper epitopes are also foreignwith respect to other IL-13 sequences from other species. However,animal pharmaceutical products are not excluded, for example canine orother veterinary species pharmaceutical products can be made in ananalogous fashion to that described for human vaccines above.

The compositions of the present invention comprise an IL-13 element andan additional element for providing T-cell help.

IL-13 Element

The IL-13 element, in its broadest form, is any sequence that is capableof driving an immune response that recognises and neutralises thebiological effects of IL-13. Preferably, the IL-13 is human IL-13.

In this context of the present invention the entire IL-13 sequences maybe used, or functional equivalent fragments thereof. Accordingly,references in this text to IL-13 sequences may encompass the entiresequence or fragments or truncates thereof.

The IL-13 element may comprise the native IL-13 sequence or a mutatedform thereof. Accordingly, the IL-13 sequence may be, for example,native human IL-13 or fragment thereof.

In an alternative embodiment of the present invention the immunogenscomprise a chimaeric IL-13 sequence that comprise substitution mutationsto swap one or more of the human sequence amino acids with theequivalent amino acids found in the same positions within the sequenceof IL-13 from another mammalian species. In the context of a humanvaccine immunogen, the object of the chimaeric sequences is to maximisethe amino acid sequence diversity between the immunogen and human nativeIL-13, whilst keeping maximal shape and conformational homology betweenthe two compositions. The chimaeric immunogen achieves this bysubstituting amino acids found in regions predicted to be masked fromthe surface. Most preferably the amino acids are substituted with aminoacids that are found in equivalent positions within an IL-13 sequencefrom another mammalian species. In this way, sequence diversity isachieved with minimal alteration to the overall shape/configuration ofthe immunogen.

In one aspect of the present invention, there is provided a human IL-13immunogen that comprises substitution mutations in areas that areassociated with alpha helical regions, which substitutions involveswapping the human amino acid with the amino acid that appears in thesame position within the IL-13 sequence of a different mammalianspecies.

Most preferably, there are substitution mutations in a plurality ofsites within the IL-13 sequence, wherein at least two or more of themutation sites comprise a substitution involving amino acids taken fromdifferent non-human mammalian species, more preferably the substitutionsinvolve amino acids taken from 3 or more different non-human mammalianspecies, and most preferably the substitutions involve amino acids takenfrom 4 or more different non-human mammalian species.

Preferably, the substitutions do not occur in at least six of the areasof high interspecies conservation: 3PVP (SEQ ID NO. 30), 12ELIEEL (SEQID NO. 31), 19NITQ (SEQ ID NO. 32), 28LCN (SEQ ID NO. 33), 32SMVWS (SEQID NO. 34), 50SL (SEQ ID NO. 35), 60AI (SEQ ID NO. 36), 64TQ (SEQ ID NO.37), 87DTKIEVA (SEQ ID NO. 38), 99LL (SEQ ID NO. 39), 106LF (SEQ ID NO.40).

The preferred IL-13 element of the immunogens of the present inventionare human chimaeric IL-13 sequences which have a similar conformationalshape to native human IL-13 whilst having sufficient amino acid sequencediversity to enhance its immunogenicity when administered to a human,characterised in that the chimaeric IL-13 immunogen has the sequence ofhuman IL-13 comprising:

(a) substitution mutations in at least two of the following alphahelical regions: PSTALRELIEELVNIT (SEQ ID NO. 41), MYCAALESLI (SEQ IDNO. 42), KTQRMLSGF (SEQ ID NO. 43) or AQFVKDLLLHLKKLFRE (SEQ ID NO. 44),

(b) comprises in unmutated form at least six of the following regions ofhigh inter-species conservation 3PVP (SEQ ID NO. 30), 12ELIEEL (SEQ IDNO. 31), 19NITQ (SEQ ID NO. 32), 28LCN (SEQ ID NO. 33), 32SMVWS (SEQ IDNO. 34), 50SL (SEQ ID NO. 35), 60AI (SEQ ID NO. 36), 64TQ (SEQ ID NO.37), 87DTKIEVA (SEQ ID NO. 38), 99LL (SEQ ID NO. 39), 106LF (SEQ ID NO.40), and

(c) optionally comprises a mutation in any of the remaining amino acids,

wherein any substitution performed in steps a, b or c is a structurallyconservative substitution.

The numerical prefix to the amino acids listed, refers to the positionalnumber of the amino acid sequence in the mature form of human IL-13,wherein the first residue “G” is assigned the number 2.

In the context of step (a) of the above chimaeric IL-13 element,preferably at least two, more preferably at least three and mostpreferably all four alpha helical regions comprise at least onesubstitution mutation. In the context of step (b) preferably at least 7,more preferably at least 8, more preferably at least 9, more preferablyat least 10, and most preferably all 11 of the regions are unmutated.

Preferably greater than 50% of these substitutions or mutations in theabove chimaeric IL-13 element, comprise amino acids taken fromequivalent positions within the IL-13 sequence of a non-human. Morepreferably more than 60, or 70, or 80 percent of the substitutionscomprise amino acids taken from equivalent positions within the IL-13sequence of a non-human mammal. Most preferably, each substitution ormutation comprise amino acids taken from equivalent positions within theIL-13 sequence of a non-human mammal.

Again in the context of the chimaeric human IL-13 element, preferablygreater than 50% of these substitutions or mutations occur in regions ofhuman IL-13 which are predicted to be alpha helical in configuration.More preferably more than 60, or 70, or 80 percent of the substitutionsor mutations occur in regions of human IL-13 which are predicted to bealpha helical in configuration. Most preferably, each substitution ormutation occurs in regions of human IL-13 which are predicted to bealpha helical in configuration.

Again in the context of the chimaeric human IL-13 elements, preferablythe human IL-13 sequence comprises between 2 and 20 substitutions, morepreferably between 6 and 15 substitutions and most preferably 13substitutions.

In the case of a human IL-13 vaccine, the IL-13 immunogen could be basedon an orthologous IL-13 sequence (such as the murine IL-13 sequence)wherein the murine B-cell epitopes (surface exposed regions) aresubstituted for the equivalent human sequences. In this embodiment themurine “backbone” will provide foreign T-cell epitopes, in addition tothe supplemental promiscuous T-cell epitopes (such as P2 or P30) whichare added either at the termini or within the chimaera sequence.

A preferred chimaeric human IL-13 immunogen comprises the sequence ofhuman IL-13, wherein the amino acid sequence comprises conservativesubstitutions, or substitutions characteristic of amino acids present atequivalent positions within the IL-13 sequence of a non-human species,present in at least six of the following 13 positions 8T, 11R, 18V, 49E,62K, 66M, 69G, 84H, 97K, 101L, 105K, 109E, 111R. Most preferably such achimaeric human IL-13 immunogen comprises at least 6, and preferrablyall, of the following substitutions: Position Substitution Species 8T->S Synthetic 11 R->K pig, cow, dog, mouse, gerbil, cyno, rhesus,marmoset. 18 V->A Synthetic 49 E->D cow, mouse, gerbil. 62 K->R cow,dog, mouse, rat. 66 M->I Mouse, gerbil, rat. 69 G->A Cow, pig, dog 84H->R Dog, rhesus, cyno 97 K->T Mouse 101 L->V Cyno, rhesus 105 K->RSynthetic 109 E->Q Marmoset 111 R->T Marmoset

The chimaeric IL-13 that comprises each of these listed substitutions isa preferred IL-13 element (Immunogen 1, SEQ ID NO. 10) and is shown inFIG. 5. Other highly preferred IL-13 elements are Immunogen 11 (SEQ IDNO. 20, see FIG. 15), Immunogen 12 (SEQ ID NO. 21, see FIG. 16) andImmunogen 13 (SEQ ID NO. 22, see FIG. 17).

The IL-13 element may also optionally further comprise a mutation thatabolishes the biological activity of the immunogen. The followingsubstitutions can be used to inactivate human IL13 bioactivity: E 12 toI, S, or Y; E12 to K; R 65 to D; S 68 to D; R 108 to D.

In certain aspects of the present invention immunogenic fragments of thenative IL-13 sequence may be used, for example in the presentation ofimmunogenic peptides in Hepatitis B core particles or in the context ofchimaeric immunogens described above. In these contexts immunogenicfragments of the human IL-13 sequences preferably contain the B-cellepitopes in the human IL-13 sequence, and preferably at least one ormore of the following short sequences: GPVPPSTA (SEQ ID NO. 45)ITQNQKAPLCNGSMVWSINLTAGM (SEQ ID NO. 46) INVSGCS (SEQ ID NO. 47)FCPHKVSAGQFSSLHVRDT (SEQ ID NO. 48) LHLKKLFREGRFN (SEQ ID NO. 49)

The polypeptide of the invention may be further modified by mutation,for example substitution, insertion or deletion of amino-acids in orderto add desirable properties (such as the addition of a sequence tag thatfacilitates purification or increase immunogenicity) or removeundesirable properties (such as an unwanted agonistic activity at areceptor) or transmembrane domains. In particular the present inventionspecifically contemplates fusion partners that ease purification such aspoly histidine tags or GST expression partners that enhance expression.A preferred tag or expression partner is immunoglobulin FC of human IgG1fused to the C-terminus of the IL-13 molecule.

Other mutations, outside of those regions that are to be left unmutateddue to their high level of conservation between species, may occur inthe IL-13 sequence. Preferably such mutations are conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5),asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. These are preferred conservative substitutions.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

Element to Provide T-Cell Help.

Associated with the IL-13 element to make the immunogens of the presentinvention, are elements that provide foreign T-cell help. Mostpreferably the T-cell helper epitopes are foreign to human sequences,but also foreign with respect to any IL-13 sequences from non-humanmammals. Preferably the T-cell helper epitopes used are small and areadded to the IL-13 sequence by an addition or substitution event within,or at the terminal ends of, the IL-13 sequence by synthetic, recombinantor molecular biological means. Alternatively the T-cell helper epitopesmay be added via chemical coupling of the IL-13 polypeptide to a carrierprotein comprising the T-cell helper epitopes. The IL-13 sequences, orfunctionally equivalent fragments thereof, may also be associated withthe T-cell helper epitopes in a fusion protein, wherein the two arerecombinantly manufactured together, for example a Hepatitis B coreprotein incorporating IL-13 sequences.

In the aspects of the present invention where small T-cell helperepitopes are used, a “foreign T-cell helper epitope” or “T-cell epitope”is a peptide which is able to bind to an MHC II molecule and stimulatesT-cells in an animal species. Preferred foreign T-cell epitopes arepromiscuous epitopes, ie. epitopes that bind multiple different MHCclass II molecules in an animal species or population (Panina-Bordignonet al, Eur. J. Immunol. 1989, 19:2237-2242; Reece et al, J. Immunol.1993, 151:6175-6184; WO 95/07707).

In order for the immunogens of the present invention to be clinicallyeffective in a complex outbred human population, it may be advantageousto include several foreign T-cell epitopes. Promiscuous epitopes mayalso be another way of achieving this same effect, including naturallyoccurring human T-cell epitopes such as those from tetanus toxoid (e.g.the P2 and P30 epitopes, diphtheria toxoid, influenza virushaemagluttinin (HA), and P. falciparum CS antigen. The most preferredT-cell epitopes for use in the present invention are P2 and P30 fromtetanus toxoid

A number of promiscuous T-cell epitopes have been described in theliterature, including: WO 98/23635; Southwood et al., 1998, J. Immunol.,160: 3363-3373; Sinigaglia et al., 1988, Nature, 336: 778-780; Rammenseeet al., 1995, Immunogenetics, 41: 4, 178-228; Chicz et al., 1993, J.Exp. Med., 178:27-47; Hammer et al., 1993, Cell 74:197-203; and Falk etal., 1994, Immunogenetics, 39: 230-242. The promiscuous T-cell epitopecan also be an artificial sequence such as “PADRE” (WO 95/07707).

The heterologous T-cell epitope is preferably selected from the group ofepitopes that will bind to a number of individuals expressing more thanone MHC II molecules in humans. For example, epitopes that arespecifically contemplated are P2 and P30 epitopes from tetanus toxoid,Panina-Bordignon Eur. J. Immunol 19 (12), 2237 (1989). In a preferredembodiment the heterologous T-cell epitope is P2 or P30 from Tetanustoxin.

The P2 epitope has the sequence QYIKANSKFIGITE (SEQ ID NO. 50) andcorresponds to amino acids 830-843 of the Tetanus toxin.

The P30 epitope (residues 947-967 of Tetanus Toxin) has the sequenceFNNFTVSFWLRVPKVSASHLE (SEQ ID NO. 51). The FNNFTV (SEQ ID NO. 52)sequence may optionally be deleted. Other universal T epitopes can bederived from the circumsporozoite protein from Plasmodium falciparum—inparticular the region 378-398 having the sequence DIEKKIAKMEKASSVFNVVNS(SEQ ID NO. 53; Alexander J, (1994) Immunity 1 (9), p 751-761).

Another epitope is derived from Measles virus fusion protein at residue288-302 having the sequence LSEIKGVIVHRLEGV (SEQ ID NO. 54; Partidos CD, 1990, J. Gen. Virol 71(9) 2099-2105).

Yet another epitope is derived from hepatitis B virus surface antigen,in particular amino acids, having the sequence FFLLTRILTIPQSLD (SEQ IDNO. 55).

Another set of epitopes is derived from diphteria toxin. Four of thesepeptides (amino acids 271-290, 321-340, 331-350,351-370) map within theT domain of fragment B of the toxin, and the remaining 2 map in the Rdomain (411-430, 431-450): PVFAGANYAAWAVNVAQVI (SEQ ID NO. 56)VHHNTEEIVAQSIALSSLMV (SEQ ID NO. 57) QSIALSSLMVAQAIPLVGEL (SEQ ID NO.58) VDIGFAAYNFVESII NLFQV (SEQ ID NO. 59) QGESGHDIKITAENTPLPIA (SEQ IDNO. 60) GVLLPTIPGKLDVNKSKTHI (SEQ ID NO. 61)(Raju R., Navaneetham D., Okita D., Diethelm-Okita B., McCormick D.,Conti-Fine B. M. (1995) Eur. J. Immunol. 25: 3207-14.)

A particularly preferred element to provide T-cell help, is a fusionpartner called “CPC” (clyta-P2-clyta) which is disclosed inPCT/EP03/06096.

Most preferably the foreign T-cell helper epitopes are “foreign” in thatthey are not tolerated by the host immune system, and also in that theyare not sequences that are derived or selected from any IL-13 sequencefrom another species (non-vaccinee).

In the aspect of the present invention where native self IL-13 iscoupled to a T-helper epitope bearing immunogenic carrier, theconjugation can be carried out in a manner well known in the art. Thus,for example, for direct covalent coupling it is possible to utilise acarbodiimide, glutaraldehyde or (N-[γ-maleimidobutyryloxy]succinimideester, utilising common commercially available heterobifunctionallinkers such as CDAP and SPDP (using manufacturers instructions). Afterthe coupling reaction, the immunogen can easily be isolated and purifiedby means of a dialysis method, a gel filtration method, a fractionationmethod etc.

The types of carriers used in the immunogens of the present inventionwill be readily known to the man skilled in the art. A non-exhaustivelist of carriers which may be used in the present invention include:Keyhole limpet Haemocyanin (KLH), serum albumins such as bovine serumalbumin (BSA), inactivated bacterial toxins such as tetanus or diptheriatoxins (TT and DT), or recombinant fragments thereof (for example,Domain 1 of Fragment C of TT, or the translocation domain of DT), or thepurified protein derivative of tuberculin (PPD). Alternatively the IL-13may be directly conjugated to liposome carriers, which may additionallycomprise immunogens capable of providing T-cell help. Preferably theratio of IL-13 to carrier molecules is in the order of 1:1 to 20:1, andpreferably each carrier should carry between 3-15 IL-13 molecules.

In an embodiment of the invention a preferred carrier is Protein D fromHaemophilus influenzae (EP 0 594 610 B1). Protein D is an IgD-bindingprotein from Haemophilus influenzae and has been patented by Forsgren(WO 91/18926, granted EP 0 594 610 B1). In some circumstances, forexample in recombinant immunogen expression systems it may be desirableto use fragments of protein D, for example Protein D ⅓^(rd) (comprisingthe N-terminal 100-110 amino acids of protein D (GB 9717953.5)).

Another preferred method of presenting the IL-13, or immunogenicfragments thereof, is in the context of a recombinant fusion molecule.For example, EP 0 421 635 B describes the use of chimaeric hepadnaviruscore antigen particles to present foreign peptide sequences in avirus-like particle. As such, immunogens of the present invention maycomprise IL-13 presented in chimaeric particles consisting of hepatitisB core antigen. Additionally, the recombinant fusion proteins maycomprise IL-13 and a carrier protein, such as NS1 of the influenzavirus. For any recombinantly expressed protein which forms part of thepresent invention, the nucleic acid which encodes said immunogen alsoforms an aspect of the present invention.

Preferred Immunogens

In the sections above, preferred definitions of the IL-13 element andthe element to provide T-cell help have been described. For thecompositions of the present invention, it is intended that this documentdiscloses each individual preferred element from the IL-13 elementsection in combination with each individual preferred element from theelement to provide T-cell help section. Particularly preferred arecombinations of Immunogens 1, 11, 12 or 13, and a carrier protein orpromiscuous T-cell helper epitope. Preferred carrier protein orpromiscuous T-cell helper epitopes include Protein D, CPC, P2 or P30.

Specifically disclosed preferred combinations of elements to formpreferred immunogens are listed herebelow.

When the IL-13 element is native human IL-13, and the element thatprovides T-cell help is a promiscuous T-cell epitope, preferred examplesinclude: Immunogen 2 (see FIG. 6, SEQ ID NO. 11), which comprises humanIL-13 with P30 inserted (underlined) into the protein (substituted forthe looped region between alpha helices C and D of human IL13).

Immunogen 3 (FIG. 7, SEQ ID NO. 12) is a Human IL-13 immunogen withN-terminal P30.

Immunogen 4 (FIG. 8, SEQ ID NO. 13) is a murine IL-13 with p30 insertedinto the protein (substituted for the looped region between alphahelices C and D of mouse IL13) this is an example of a mouse version ofan IL13 autovaccine. The p30 region is underlined.

Immunogen 5 (FIG. 9, SEQ ID NO. 14) is a murine IL13 with p30 at theN-terminus. This is an example of a mouse version of an IL13autovaccine. The p30 region is underlined and is positioned at theN-terminus of the mature mouse IL13 protein sequence.

Specific examples where the IL-13 element is provided as a chimaericIL-13 immunogen include:

Immunogen 6 (FIG. 10, SEQ ID NO. 15). This is an example of a mouseversion of this form of the vaccine, where there is “human backbone”sequence grafted to murine B-cell surface exposed epitopes, with P30added at the N-terminus.

Other preferred immunogens are based on a human chimaeric IL-13“Immunogen 1” (SEQ ID NO. 10). For example, Immunogen 1 is preferablyN-terminally fused to the carrier “CPC” to form Immunogen 7 (SEQ ID NO.16, see FIG. 11), or N-terminally fused to protein D (the protein Dfusion region corresponds to amino acids S20 to T127 inclusive, of H.influenzae protein D sequence (nb, the DNA sequence encoding the proteinD is codon optimised) for Immunogen 8 (SEQ ID NO. 17, see FIG. 12); orN-terminally fused to P30 to give Immunogen 9 (SEQ ID NO.18, see FIG.13). Immunogen 9 preferably further comprises the E121 mutation toabrogate any IL-13 biological activity, to give Immunogen 10 (SEQ ID NO.19, see FIG. 14).

The protein and DNA sequences shown for Immunogens 1 to 10 are shownwithout the amino acid or DNA sequence for the signal sequence requiredto drive secretion of the product from the cell. Preferably, therefore,the sequences further are further provided with a signal sequence. Inthe context of DNA vaccines it is specifically preferred that the signalsequence is a non-human derived sequence that comprises a T-cellepitope, to further provide T-cell help. None of the disclosed preferredsequences have a stop codon as it may be useful to express them fused toother molecules eg immunoglobulin Fc, 6His to facilitate production orpurification.

The numbering system used herein conforms with normal practice in thefield of IL-13, in that the G in “GPVPP” is referred to as residue 2,and the remaining amino acids are numbered accordingly.

Methods of Designing a Vaccine

In an important aspect of the present invention, there is provided amethod of designing a vaccine for the treatment of an individualsuffering from or susceptible to a disease that is susceptible totreatment by neutralisation of the activity of IL-13. Such diseasesinclude COPD, asthma and atopic disorders such as hayfever, contactallergies and atopic dermatitis.

The methods disclosed herein comprise two major steps: 1. Designing achimaeric IL-13 immunogen, and 2. Associating to the IL-13 immunogen, asource of T-cell epitopes that are foreign with respect to any humanself epitope and also foreign with respect to any mammalian IL-13sequence.

In this context the method comprises:

(a) taking the sequence of human IL-13 and identifying regions that arepredicted to form an alpha helical structure, and

(b) mutating the sequence of human IL-13 within these alpha helicalregions to substitute amino acids from the human sequence with aminoacids that are either a conservative substitution or are found inequivalent positions within the IL-13 sequence of a different species,and

c) attaching or inserting a source of T-cell epitopes that are foreignwith respect to any human self epitope and also foreign with respect toany mammalian IL-13 sequence.

As a general principle the object of the method is to design a chimaericsequence having a maximum sequence diversity between the immunogen andhuman native IL-13, whilst keeping maximal shape and conformationalhomology between the two compositions. The chimaeric immunogen achievesthis by substituting amino acids found in regions predicted to be maskedfrom the surface. Most preferably the amino acids are substituted withamino acids that are found in equivalent positions within an IL-13sequence from another mammalian species. In this way, sequence diversityis achieved with minimal alteration to the overall shape/configurationof the immunogen.

Therefore, the preferred methods of designing a chimaeric IL-13immunogen comprise the following steps:

1. Collect together IL13 sequences from other and align using tool suchas Clustal or Pileup,

2. Avoid mutations within positions which are essentially invariantacross the collection. Particularly 3PVP (SEQ ID NO. 30), 12ELIEEL (SEQID NO. 31), 19NITQ (SEQ ID NO. 32), 28LCN (SEQ ID NO. 33), 32SMVWS (SEQID NO. 34), 50SL (SEQ ID NO. 35), 60AI (SEQ ID NO. 36), 64TQ (SEQ ID NO.37), 87DTKIEVA (SEQ ID NO. 38), 99LL (SEQ ID NO. 39), 106LF (SEQ ID NO.40),

3. In the remaining sequence, favour mutations that occur in the helicalregions (PSTALRELIEELVNIT, (SEQ ID NO. 41) MYCAALESLI, (SEQ ID NO. 42)KTQRMLSGF (SEQ ID NO. 43) or AQFVKDLLLHLKKLFRE, (SEQ ID NO. 44)4. Regions not specified in 3 or 4 may optionally contain mutations.5. Mutations are selected by considering either residues which occur inother species IL13 molecules at orthologous positions, or those whichare chemically conservative.

Molecular modelling may be used to select particularly favourablesubstitutions which have a low probability of affecting the overallshape of the molecule by steric clashes etc.

Accordingly there is provided a method for the manufacture of a humanchimaeric IL-13 immunogen which has a similar conformational shape tonative human IL-13 whilst having sufficient amino acid sequencediversity to enhance its immunogenicity when administered to a human,the method comprising the following steps:

(a) taking the sequence of human IL-13 and performing at least onesubstitution mutation in at least two of the following alpha helicalregions: PSTALRELIEELVNIT (SEQ ID NO. 41), MYCAALESLI (SEQ ID NO. 42),KTQRMLSGF (SEQ ID NO. 43) or AQFVKDLLLHLKKLFRE (SEQ ID NO. 44),

(b) preserving at least six of the following regions of highinter-species conservation 3PVP (SEQ ID NO. 30), 12ELIEEL (SEQ ID NO.31), 19NITQ (SEQ ID NO. 32), 28LCN (SEQ ID NO. 33), 32SMVWS (SEQ ID NO.34), 50SL (SEQ ID NO. 35), 60AI (SEQ ID NO. 36), 64TQ (SEQ ID NO. 37),87DTKIEVA (SEQ ID NO. 38), 99LL (SEQ ID NO. 39), 106LF (SEQ ID NO. 40),

(c) optionally mutating any of the remaining amino acids, and

(d) attaching a source of T-cell epitopes that are foreign with respectto any human self epitope and also foreign with respect to any mammalianIL-13 sequence,

characterised in that any substitution performed in steps a, b or c is astructurally conservative substitution.

In the context of step (a) preferably at least two, more preferably atleast three and most preferably all four alpha helical regions compriseat least one substitution mutation. In the context of step (b)preferably at least 7, more preferably at least 8, more preferably atleast 9, more preferably at least 10, and most preferably all 11 of theregions are unmutated.

Alternatively there is provided, a method for the manufacture of a humanchimaeric IL-13 immunogen which has a similar conformational shape tonative human IL-13 whilst having sufficient amino acid sequencediversity to enhance its immunogenicity when administered to a human,the method comprising the following steps:

(a) aligning IL-13 amino acid sequences from different species,

(b) identifying regions of high variability and high conservation,

(c) taking the sequence of human IL-13 and mutating it in the areas ofhigh variability to substitute amino acids from the human sequence withamino acids that are either a conservative substitution or are found inequivalent positions within the IL-13 sequence of a different species,and

(d) attaching a source of T-cell epitopes that are foreign with respectto any human self epitope and also foreign with respect to any mammalianIL-13 sequence,

In a related aspect of the present invention, there is also provided amethod for the manufacture of a human chimaeric IL-13 immunogencomprising the following steps:

(a) aligning IL-13 amino acid sequences from different species,

(b) identifying regions of high variability and high conservation,

(c) taking the sequence of human IL-13 and mutating it in the areas ofhigh conservation to substitute amino acids from the human sequence withamino acids that are either a conservative substitution or are found inequivalent positions within the IL-13 sequence of a different species,and

(d) attaching a source of T-cell epitopes that are foreign with respectto any human self epitope and also foreign with respect to any mammalianIL-13 sequence,

In all of these methods, preferably greater than 50% of thesesubstitutions or mutations comprise amino acids taken from equivalentpositions within the IL-13 sequence of a non-human. More preferably morethan 60, or 70, or 80 percent of the substitutions comprise amino acidstaken from equivalent positions within the IL-13 sequence of a non-humanmammal. Most preferably, each substitution or mutation comprise aminoacids taken from equivalent positions within the IL-13 sequence of anon-human mammal.

Again in the context of the methods for designing chimaeric humanimmunogens, preferably greater than 50% of these substitutions ormutations occur in regions of human IL-13 which are predicted to bealpha helical in configuration. More preferably more than 60, or 70, or80 percent of the substitutions or mutations occur in regions of humanIL-13 which are predicted to be alpha helical in configuration. Mostpreferably, each substitution or mutation occurs in regions of humanIL-13 which are predicted to be alpha helical in configuration.

Again in the context of the methods of designing chimaeric humanimmunogens, preferably the immunogen comprises between 2 and 20substitutions, more preferably between 6 and 15 substitutions, and mostpreferably 13 substitutions.

Most preferably, in all of these above methods there are substitutionmutations in a plurality of sites within the IL-13 sequence, wherein atleast two or more of the mutation sites comprise a substitutioninvolving amino acids taken from different non-human mammalian species,more preferably the substitutions involve amino acids taken from 3 ormore different non-human mammalian species, and most preferably thesubstitutions involve amino acids taken from 4 or more differentnon-human mammalian species.

The present invention also provides an immunogen that is derivable fromany of the above methods, which immunogens are immunogenic, whenformulated in an appropriate manner for a vaccine, in a human vaccinee.

The successful design of a polypeptide according to the presentinvention can be verified for example by administering the resultingpolypeptide in a self-context in an appropriate vaccination regime, andobserving that antibodies capable of binding the protein are induced.This binding may be assessed through use of ELISA techniques employingrecombinant or purified native protein, or through bioassays examiningthe effect of the protein on a sensitive cell or tissue. A particularlyfavoured assessment is to observe a phenomenon causally related toactivity of the protein in the intact host, and to determine whether thepresence of antibodies induced by the methods of the invention modulatethat phenomenon. Thus a protein of the present invention will be able toraise antibodies to the native antigen in the species from which thenative protein is derived.

The most successful of designs will be able to be used in an experiment,such as that described in Example 2 herein, and induce anti-IL-13neutralising immune responses that exceed ED100 in at least 50% of thevaccinated individuals.

Vaccine Formulations

The vaccine formulations of the present invention may be in the form ofa protein based vaccine, most often formulated together with anadjuvant, or alternatively the vaccine may take the form of a DNA orpolynucleotide vaccine.

The polypeptide immunogens of the invention may be encoded bypolynucleotides of the invention. A person skilled in the art willreadily be able to determine the sequence of the polynucleotide whichencodes the polypeptide by applying the genetic code. Once the requirednucleic acid sequence has been determined, the polynucleotide with thedesired sequence can be produced as described in the examples. A skilledperson will readily be able to adapt any parameters necessary, such asprimers and PCR conditions. It will also be understood by a personskilled in the art that, due to the degeneracy of the genetic code,there is potentially more than one polynucleotide which encodes apolypeptide of the invention. The polynucleotides of the presentinvention may also comprise a region which encodes a secretion signalpeptide.

The polynucleotide of the invention is typically RNA, for example mRNA,or DNA, for example genomic DNA, cDNA or synthetic DNA. Preferably thepolynucleotide is DNA. Particularly preferably it is cDNA.

The present invention further provides an expression vector, which is anucleic acid construct, comprising the polynucleotide of the invention.Additionally, the nucleic acid construct will comprise appropriateinitiators, promoters, enhancers and other elements, such as forexample, polyadenylation signals, which may be necessary, and which arepositioned in the correct orientation, in order to allow for proteinexpression within a mammalian cell.

The promoter may be a eukaryotic promoter for example a CD68 promoter,Gal1, Gal10, or NMT1 promoter, a prokaryotic promoter for example Tac,Trc, or Lac; or a viral promoter, for example the cytomegaloviruspromoter, the SV40 promoter, the polyhedrin promoter, the P10 promoter,or the respiratory syncytial virus LTR promoter. Preferably the promoteris a viral promoter. Particularly preferred is when the promoter is thecytomegalovirus immediate early promoter, optionally comprising exon 1from the HCMV IE gene.

The transcriptional regulatory elements may comprise enhancers, forexample the hepatitis B surface antigen 3′untranslated region, the CMVenhancer; introns, for example the CD68 intron, or the CMV intron A, orregulatory regions, for example the CMV 5′ untranslated region.

The polynucleotide is preferably operably linked to the promoter on thenucleic acid construct such that when the construct is inserted into amammalian cell, the polynucleotide is expressed to produce a encodedpolypeptide.

The nucleic acid construct backbone may be RNA or DNA, for exampleplasmid DNA, viral DNA, bacterial DNA, bacterial artificial chromosomeDNA, yeast artificial chromosome DNA, synthetic DNA It is also possiblefor the nucleic acid construct to be artificial nucleic acid, forexample phosphorothioate RNA or DNA. Preferably the construct is DNA.Particularly preferred is when it is plasmid DNA.

The present invention further provides a host cell comprising anexpression vector of the invention. Such cells include transient, orpreferably stable higher eukaryotic cell lines, such as mammalian cellsor insect cells, using for example a baculovirus expression system,lower eukaryotic cells, such as yeast or prokaryotic cells such asbacterial cells. Particular examples of cells which may be modified byinsertion of vectors encoding for a polypeptide according to theinvention include mammalian HEK293T, CHO, HeLa, NS0 and COS cells.Preferably the cell line selected will be one which is not only stable,but also allows for mature glycosylation of a polypeptide. Expressionmay be achieved in transformed oocytes. A polypeptide of the inventionmay be expressed in cells of a transgenic non-human animal, preferably amouse or expressed into the milk of larger mammals, such as goats, sheepand cows. A transgenic non-human animal expressing a polypeptide of theinvention is included within the scope of the invention. A polypeptideof the invention may also be expressed in Xenopus laevis oocytes.

The present invention also includes pharmaceutical or vaccinecompositions, which comprise a therapeutically effective amount ofpolynucleotide or nucleic acid construct or polypeptide of theinvention, optionally in combination with a pharmaceutically acceptablecarrier, preferably in combination with a pharmaceutically acceptableexcipient such as phosphate buffered saline (PBS), saline, dextrose,water, glycerol, ethanol, liposomes or combinations thereof. The vaccinecomposition may alternatively comprise a therapeutically effectiveamount of a nucleic acid construct of the invention, formulated ontometal beads, preferably gold beads. The vaccine composition of theinvention may also comprise an adjuvant, such as, for example, in anembodiment, imiquimod, tucaresol or aluminium salts.

Preferably the adjuvant is administered at the same time as theimmunogens of the present invention, and in preferred embodiments areformulated together. Such adjuvant agents contemplated by the inventioninclude, but this list is by no means exhaustive and does not precludeother agents: synthetic imidazoquinolines such as imiquimod [S-26308,R-837], (Harrison, et al. ‘Reduction of recurrent HSV disease usingimiquimod alone or combined with a glycoprotein vaccine’, Vaccine 19:1820-1826, (2001)); and resiquimod [S-28463, R-848] (Vasilakos, et al.‘Adjuvant activites of immune response modifier R-848: Comparison withCpG ODN’, Cellular immunology 204: 64-74 (2000).), Schiff bases ofcarbonyls and amines that are constitutively expressed on antigenpresenting cell and T-cell surfaces, such as tucaresol (Rhodes, J. etal. ‘Therapeutic potentiation of the immune system by costimulatorySchiff-base-forming drugs’, Nature 377: 71-75 (1995)), cytokine,chemokine and co-stimulatory molecules, Th1 inducers such as interferongamma, IL-2, IL-12, IL-15 and IL-18, Th2 inducers such as IL-4, IL-5,IL-6, IL-10 and other chemokine and co-stimulatory genes such as MCP-1,MIP-1 alpha, MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD40L, otherimmunostimulatory targeting ligands such as CTLA-4 and L-selectin,apoptosis stimulating proteins and peptides such as Fas, (49), syntheticlipid based adjuvants, such as vaxfectin, (Reyes et al., ‘Vaxfectinenhances antigen specific antibody titres and maintains Th1 type immuneresponses to plasmid DNA immunization’, Vaccine 19: 3778-3786) squalene,alpha-tocopherol, polysorbate 80, DOPC and cholesterol, endotoxin,[LPS], Beutler, B., ‘Endotoxin, ‘Toll-like receptor 4, and the afferentlimb of innate immunity’, Current Opinion in Microbiology 3: 23-30(2000)); CpG oligo- and di-nucleotides, Sato, Y. et al.,‘Immunostimulatory DNA sequences necessary for effective intradermalgene immunization’, Science 273 (5273): 352-354 (1996). Hemmi, H. etal., ‘A Toll-like receptor recognizes bacterial DNA’, Nature 408:740-745, (2000) and other potential ligands that trigger Toll receptorsto produce Th1-inducing cytokines, such as synthetic Mycobacteriallipoproteins, Mycobacterial protein p19, peptidoglycan, teichoic acidand lipid A.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a Lipid A derivative such asmonophosphoryl lipid A, or preferably 3-de-O-acylated monophosphoryllipid A. MPL® adjuvants are available from Corixa Corporation (Seattle,Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034and 4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) also induce a predominantly Th1 response.Such oligonucleotides are well known and are described, for example, inWO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Satoet al., Science 273:352, 1996. Another preferred adjuvant comprises asaponin, such as Quil A, or derivatives thereof, including QS21 and QS7(Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin;or Gypsophila or Chenopodium quinoa saponins.

In particular, the adjuvant comprises an immunostimulatory CpGoligonucleotide, such as disclosed in (WO96102555). Typicalimmunostimulatory oligonucleotides will be between 8-100 bases in lengthand comprises the general formula X₁CpGX₂ where X₁ and X₂ are nucleotidebases, and the C and G are unmethylated.

The preferred oligonucleotides for use in vaccines of the presentinvention preferably contain two or more dinucleotide CpG motifspreferably separated by at least three, more preferably at least six ormore nucleotides. The oligonucleotides of the present invention aretypically deoxynucleotides. In a preferred embodiment theinternucleotide in the oligonucleotide is phosphorodithioate, or morepreferably a phosphorothioate bond, although phosphodiester and otherinternucleotide bonds are within the scope of the invention includingoligonucleotides with mixed internucleotide linkages. e.g. mixedphosphorothioate/phophodiesters. Other internucleotide bonds whichstabilise the oligonucleotide may be used. Methods for producingphosphorothioate oligonucleotides or phosphorodithioate are described inU.S. Pat. No. 5,666,153, U.S. Pat. No. 5,278,302 and WO95/26204.

Examples of preferred oligonucleotides have the following sequences. Thesequences preferably contain phosphorothioate modified internucleotidelinkages. (SEQ ID NO. 62) OLIGO 1: TCC ATG ACG TTC CTG ACG TT (CpG 1826)(SEQ ID NO. 63) OLIGO 2: TCT CCC AGC GTG CGC CAT (CpG 1758) (SEQ ID NO.64) OLIGO 3: ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG (SEQ ID NO. 65)OLIGO 4: TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006) (SEQ ID NO. 66)OLIGO 5: TCC ATG ACG TTC CTG ATG CT (CpG 1668)Alternative CpG oligonucleotides may comprise the preferred sequencesabove in that they have inconsequential deletions or additions thereto.

The CpG oligonucleotides utilised in the present invention may besynthesized by any method known in the art (eg EP 468520). Conveniently,such oligonucleotides may be synthesized utilising an automatedsynthesizer. An adjuvant formulation for use in mice and containing CpGoligonucleotide can be purchased from Qiagen under the trade name“ImmunEasy”. Preferably the adjuvant is one of the CpG's defines asOLIGO's 1, 2, 3, 4 or 5 adsorbed to aluminium hydroxide at anapproximate 1:1 ratio weight/weight. OLIGO 4 is preferred for use inhumans.

Preferably the CpG is in combination with a saponin, such as QS21, asdescribed in WO 00/62800 and WO 00/09159 the contents of both of whichis encorporated herein by reference.

Methods of Treatment

The present invention provides novel treatments for atopic diseases,comprising an immunogen that is capable of generating an immune responsein a vaccinee against IL-13. Most notably the present invention providesa method of treating an individual suffering from or being susceptibleto COPD, asthma or atopic dermatitis, comprising administering to thatindividual a vaccine according to the present invention, and therebyraising in that individual a serum neutralising anti-IL-13 immuneresponse and thereby ameliorating or abrogating the symptoms of COPD,asthma or atopic dermatitis.

Also provided by the present invention is the use of the immunogens ofthe present invention in the manufacture of a medicament for thetreatment asthma. Also provided is a method of treatment of asthmacomprising the administration to an individual in need thereof of apharmaceutical composition or vaccine as described herein.

Preferably the pharmaceutical composition is a vaccine that raises animmune response against IL-13. The immune response raised is preferablyan antibody response, most preferably an IL-13 neutralising antibodyresponse.

The invention also provides:

an expression vector which comprises a polynucleotide of the inventionand which is capable of expressing a polypeptide of the invention;

a host cell comprising an expression vector of the invention;

a method of producing a polypeptide of the invention which methodcomprises maintaining a host cell of the invention under conditionssuitable for obtaining expression of the polypeptide and isolating thesaid polypeptide:

a vaccine composition comprising a polypeptide or polynucleotide of theinvention and a pharmaceutically acceptable carrier.

The methods of treatment of the present invention provide a method oftreatment of asthma comprising one or more of the following clinicaleffects:

1. A reduction in airway hyper-responsiveness (AHR)

2. A reduction in mucus hyper-secretion and goblet cell metaplasia

3. A reduction in sub-epithelial fibrosis of the airways

4. A reduction in eosinophil levels

5. A reduction in the requirement for the use of inhaled corticosteroids(ICS) would also be a feature of successfull treatment using an IL13autovaccine.

The compositions of the present invention may be used for bothprophylaxis and therapy. The present invention provides a polypeptide ora polynucleotide according to the invention for use in medicine. Theinvention further provides the use of a polypeptide or a polynucleotideof the invention in the manufacture of a medicament for the treatment ofallergies, respiratory ailments such as asthma and COPD,helminth-infection related disorders, fibrosis or cirrhosis of theliver.

The present invention also provides a method of vaccinating whichcomprises administering an effective amount of a vaccine composition ofthe invention to a patient and provoking an immune response to thevaccine composition.

The present invention also provides vaccine compositions as describedherein for use in vaccination of a mammal against IL-13 mediateddisorders such as allergies, respiratory ailments, helminth-infectionrelated disorders, fibrosis and cirrhosis of the liver. A vaccinecomposition capable of directing a neutralising response to IL-13 wouldtherefore constitute a useful therapeutic for the treatment of asthma,particularly allergic asthma, in humans. It would also have applicationin the treatment of certain helminth infection-related disorders(Brombacher, 2000 Bioessays 22:646-656) and diseases where IL-13production is implicated in fibrosis (Chiaramonte et al, 1999, J ClinInv 104:777-785), such as chronic obstructive pulmonary disease (COPD)and cirrhosis of the liver.

The methods of treatment of the present invention provide a method oftreatment of atopic dermatitis comprising one or more of the followingclinical effects:

1. A reduction in skin irritation

2. A reduction in itching and scratching

3. A reduction in the requirement for conventional treatment.

4. if applicable a reduction in the requirement for the use of topicalcorticosteroids. An ideal IL13 autovaccine could potentially make ICSsteroid treatment redundant, although a reduction in the ‘frequency ofuse’ or ‘dose required’ of ICS is also envisaged as a valuable outcome.

The present invention also provides methods of treating or preventingIL-13 mediated disease, any symptoms or diseases associated therewith,comprising administering an effective amount of a protein, apolynucleotide, a vector or a pharmaceutical composition according tothe invention. Administration of a pharmaceutical composition may takethe form of one or more individual doses, for example in a “prime-boost”therapeutic vaccination regime. In certain cases the “prime” vaccinationmay be via particle mediated DNA delivery of a polynucleotide accordingto the present invention, preferably incorporated into a plasmid-derivedvector and the “boost” by administration of a recombinant viral vectorcomprising the same polynucleotide sequence, or boosting with theprotein in adjuvant. Conversly the priming may be with the viral vectoror with a protein formulation typically a protein formulated in adjuvantand the boost with a DNA vaccine of the present invention.

The present invention provides methods of generating an anti self IL-13antibody response in a host by the administration of vaccines of thepresent invention.

The vaccine compositions of the invention may be administered in avariety of manners for example via the mucosal, such as oral and nasal;pulmonary, intramuscular, subcutaneous or intradermal routes. Where theantigen is to be administered as a protein based vaccine, the vaccinewill typically be formulated with an adjuvant and may be lyophilised andresuspended in water for injection prior to use. Such compositions maybe administered to an individual as an injectable composition, forexample as a sterile aqueous dispersion, preferably isotonic. Typicallysuch compositions will be administered intra muscularly, but otherroutes of administration are possible.

One technique for intradermally administration involves particlebombardment (which is also known as ‘gene gun’ technology and isdescribed in U.S. Pat. No. 5,371,015). Proteins may be formulated withsugars to form small particles or DNA encoding the antigen may be coatedon to inert particles (such as gold beads) and are accelerated at speedssufficient to enable them to penetrate a surface of a recipient (e.g.skin), for example by means of discharge under high pressure from aprojecting device. (Particles coated with nucleic acid vaccineconstructs of the invention and protein sugar particles are within thescope of the present invention, as are devices loaded with suchparticles.) Other methods of administering the nucleic acid constructsor compositions containing said constructs directly to a recipientinclude ultrasound, electrical stimulation, electroporation andmicroseeding which is described in U.S. Pat. No. 5,697,901.

A nucleic acid construct of the present invention may also beadministered by means of specialised delivery vectors useful in genetherapy. Gene therapy approaches are discussed for example by Verme etal, Nature 1997, 389:239-242. Both viral and non-viral systems can beused. Viral based systems include retroviral, lentiviral, adenoviral,adeno-associated viral, herpes viral and vaccinia-viral based systems.Non-viral based systems include direct administration of nucleic acidsand liposome-based systems. For example, the vectors may be encapsulatedby liposomes or within polylactide co-glycolide (PLG) particles. Anucleic acid construct of the present invention may also be administeredby means of transformed host cells. Such cells include cells harvestedfrom a subject. The nucleic acid vaccine construct can be introducedinto such cells in vitro and the transformed cells can later be returnedto the subject. The nucleic acid construct of the invention mayintegrate into nucleic acid already present in a cell by homologousrecombination events. A transformed cell may, if desired, be grown up invitro and one or more of the resultant cells may be used in the presentinvention. Cells can be provided at an appropriate site in a patient byknown surgical or microsurgical techniques (e.g. grafting,micro-injection, etc.). Suitable cells include dendritic cells.

The amount of vaccine composition which is delivered will varysignificantly, depending upon the species and weight of mammal beingimmunised, the nature of the disease state being treated/protectedagainst, the vaccination protocol adopted (i.e. single administrationversus repeated doses), the route of administration and the potency anddose of the adjuvant compound chosen. Based upon these variables, amedical or veterinary practitioner will readily be able to determine theappropriate dosage level but it may be, for example, when the vaccine isa nucleic acid that the dose will be 0.5-5 μg/kg of the nucleic acidconstructs or composition containing them. In particular, the dose willvary depending on the route of administration. For example, when usingintradermal administration on gold beads, the total dosage willpreferably between 1 μg-10 ng, particularly preferably, the total dosagewill be between 10 μg and 1 ng. When the nucleic acid construct isadministered directly, the total dosage is generally higher, for examplebetween 50 μg and 1 or more milligram. The above dosages are exempla ofthe average case.

In a protein vaccine, the amount of protein in each vaccine dose isselected as an amount which induces an immunoprotective response withoutsignificant, adverse side effects in typical vaccinees. Such amount willvary depending upon which specific immunogen is employed and how it ispresented. Generally, it is expected that each dose will comprise 1-1000μg of protein, preferably 1-500 μg, preferably 1-100 μg, most preferably1 to 50 μg. An optimal amount for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in vaccinated subjects. Following an initialvaccination, subjects may receive one or several booster immunisationadequately spaced. Such a vaccine formulation may be either a priming orboosting vaccination regime; be administered systemically, for examplevia the transdermal, subcutaneous or intramuscular routes or applied toa mucosal surface via, for example, intra nasal or oral routes.

There can, of course, be individual instances where higher or lowerdosage ranges are merited, and such are within the scope of thisinvention.

It is possible for the vaccine composition to be administered on a onceoff basis or to be administered repeatedly, for example, between 1 and 7times, preferably between 1 and 4 times, at intervals between about 1day and about 18 months, preferably one month. This may be optionallyfollowed by dosing at regular intervals of between 1 and 12 months for aperiod up to the remainder of the patient's life. In an embodiment thepatient will receive the antigen in different forms in a prime boostregime. Thus for example an antigen will be first administered as a DNAbased vaccine and then subsequently administered as a protein adjuvantbase formulation. Once again, however, this treatment regime will besignificantly varied depending upon the size and species of animalconcerned, the amount of nucleic acid vaccine and/or protein compositionadministered, the route of administration, the potency and dose of anyadjuvant compounds used and other factors which would be apparent to askilled veterinary or medical practitioner.

Throughout this specification the words “comprise” and “include” orvariations such as “comprising”, “comprises”, “including”, “includes”etc., are to be construed both inclusively, that is, use of these wordswill imply the possible inclusion of integers or elements notspecifically recited and also in the exclusionary sense in that thewords could be read as “consisting”.

As described herein, the present invention relates isolated polypeptidesand isolated polynucleotides. In the context of this invention the term“isolated” is intended to convey that the polypeptide or polynucleotideis not in its native state, insofar as it has been purified at least tosome extent or has been synthetically produced, for example byrecombinant methods, or mechanical synthesis. The term “isolated”therefore includes the possibility of the polypeptides orpolynucleotides being in combination with other biological ornon-biological material, such as cells, suspensions of cells or cellfragments, proteins, peptides, expression vectors, organic or inorganicsolvents, or other materials where appropriate, but excludes thesituation where the polynucleotide is in a state as found in nature.

The present invention is exemplified, but not limited to, the followingexamples.

EXAMPLE 1 Methodology

For the methods below the following nomenclature applies:

1. The construct called mouse IL13 (mIL-13) with tetanus toxin p30epitope inserted into the protein (substituted into the looped regionbetween alpha helices C and D of mouse IL13) is referred to asmIL13p30CD.

2. The construct called mouse IL13 with p30 at the N-terminus, isreferred to as mIL13p30.

3. The construct called new chimaeric IL13 design with p30 N-terminus,is referred to as cIL13new.

IL-13 Subcloning/Modifications:

A gene (mIL13CD) encoding mIL-13 containing the p30 epitope from tetanustoxin inserted into the CD loop was prepared synthetically. Thesynthetic gene contains a 5′ KpnI restriction site and a 3′ BamHIrestriction site. This fragment was then subcloned between the Kpn I andBam HI restriction sites of pCDN which encodes DHFR (Aiyer et al, 1994).The resultant intermediate was subsequently modified by inserting an FCfusion. Site-directed insertional mutagenesis was used to preciselyinsert human IgG1 FC in frame with the 3′ end coding sequence precedingthe stop codon of IL-13 (Geisser et al 2001). This was performed in twosteps 1. IgG1 FC was amplified from a cDNA template, pCDN-FC, using thefollowing primer set, (Forward: 5′ . . .CAACTGTTTCGCCACGGCCCCTTCCTGGAGGTCCTGTTCGGTGGACCAGGATCCGAGCCCAAATCGGCCGAC. . . 3′ (SEQ ID NO 67) and Reverse: 5′ . . . CTAGGTAGTTGGTAACCGTTAACGG. . . 3′ (SEQ ID NO. 68)) in a PCR reaction catalyzed by KODproof-reading polymerase (Novagen). 2. The resultant PCR product was gelpurified and 250 ng used as a targeting fragment in a site-directedmutagenesis reaction using the QuickChange kit (Stratagene) with 50 ngmIL-13 CD-pCDN and 2.5 U PfuTurbo. The mutagenesis protocol consisted of18 Cycles of 30 s at 95° C., 30S at 55° C., and 16 minutes at 68° C. Atthe end of the mutagenesis protocol, the reaction was digested with 10 UDpn I to remove the original methylated wild-type template DNA. 1 ul ofthe final digested reaction was used to transform 100 ul Epicurianchemically competent E. coli cells (Stratagene). Recombinant clones werescreened by restriction digestion and positive clones sequence confirmedfully across the FC region using IL-13 forward and pCDN reverse primers.The final plasmid, pCDNmIL13CDFC encodes a C-terminal FC fusionseparated by a PreScission protease cleavage site for FC removal.Transcription is under control of the CMV promoter. The completesequence of the insert is shown in FIG. 18 (SEQ ID NO. 23).

pCDNmIL13p30FC was constructed in exactly the same way as describedabove for pCDNmIL13CDFC, replacing the mIL13CD synthetic gene with onewhere the p30 epitope was present at the N terminus of the matureprotein instead of being in the CD loop. The same forward and reverseprimers were used to generate the targeting fragment for site-directedinsertion of the FC region into pCDNmIL13p30. The complete sequence ofthe insert is shown in FIG. 19 (SEQ ID NO. 24)

pCDNcIL13newFC was constructed using a synthetic gene encoding thecIL13new molecule and the following forward primer (5′ . . .AACCTGTTTCGCCGCGGCCCCTTCCTGGAGGTCCTGTTCGGTGGACCAGGATCCGAGCCCAAATCGGCCGAC. . . 3′, (SEQ ID NO. 25)) and the same reverse primer described aboveto generate the targeting fragment for site-directed insertion of the FCregion into pCDNcIL13new. The complete sequence of the insert is shownin FIG. 20 (SEQ ID NO. 26)).

pCDN IL13oldFC was constructed by site-directed replacement of mIL13 CDwithin pCDNmIL13CDFC with mouse chimeric IL13 (see WO 02/070711).Site-directed replacement was performed as described for site-directedinsertion. cIL13 was PCR amplified from 6His-cIL13 using the followingprimers (Forward: 5′ 5′ . . . GTGTCTCTCCCTCTGACCCTTAGG . . . 3′ (SEQ IDNO. 27) and Reverse: 5′ . . . CAGTTGCTTTGTGTAGCTGAG CAG . . . 3′ (SEQ IDNO. 28) to generate a targeting fragment for replacement into pCDNmIL13.This generates a precise fusion to the IL-13 signal sequence encoded atthe 5′ end and the PreScission-FC region encoded at the 3′ end. Thecomplete sequence of the insert is shown in FIG. 21 (SEQ ID NO. 29).

In all of FIGS. 18 to 21, doubly underlined amino acid residues indicatethe secretion signal sequence (removed in the course of expression andsecretion from the host cell), single underlined residues, thePrecission protease site and italicised residues the Fc fusion partner.

Generation of Stable CHO E1A Clones:

Plasmids were stably expressed in a DHFR negative, E1A expressing line(CHO E1A, ACC317). Cells were resuspended at 1×10⁷ cell/ml in coldphosphate buffered sucrose, transferred to a Gene Pulser Cuvette, andelectroporated with 15 ug Not I linearized plasmid at 400 volt and 25uFd in a GenePulser (Biorad). Electroporated cells were plated in a 96well plate at 2.5×10³ viable cells per well in complete mediumcontaining 1×Nucleosides. After 48 hours the medium was exchanged withfresh medium lacking nucleosides. Cells were subsequently selected over3-4 weeks in the absence of nucleosides. Positive clones were screenedfrom the 96 well plate by monitoring FC expression from conditionedmedium using an FC-electrochemiluminescence detection protocol (Yang, etal., 1994) on an Origen analyzer (IGEN). Positive cell lines were scaledto several litres in complete medium minus nucleosides. Fermentationswere carried out at 34° C. for 10-11 days. Conditioned medium washarvested and 0.2 uM sterile filtered in preparation for FCpurification.

Purification:

Murine IL13CD/Fc was captured from CHO medium onto ProSep-A HighCapacity resin (Bioprocessing Limited). The murine IL13CD/Fc was elutedfrom the ProSep-A resin with 0.1M Glycine pH=3.0, neutralized with 1MHEPES pH=7.6, and dialyzed against 25 mM sodium phosphate 0.15M sodiumchloride pH=7 (Spectra/Por® 7 membrane, MWCO:8000). Overall yield was644 mg murine IL13CD/Fc from 3.8 liter CHO medium. Other IL13/Fc fusionproteins were prepared similarly.

Before use in vaccination studies, the Fc portions of these moleculeswere cleaved off using Precission protease and removed. The resultingvaccine preparations comprise essentially those amino acid residuesindicated in FIGS. 18 to 21 by plain text (ie neither underlined noritalicised).

REFERENCES

-   Aiyer, N, Baker, E, Wu, H-L, Nambi, P, Edwards, R M, Trill, J J,    Ellis, C, Bergsma, D J. (1994): Human ATI receptor is a single copy    gene: characterization in a stable cell line. Molecular and Cellular    Biochemistry 131:75-86.-   Geiser, M, Cebe, R, Drewello, D, and Schmitz, R (2001): Integration    of PCR Fragments at Any Specific Site within Cloning Vectors without    the Use of Restriction Enzymes and DNA Ligase. Biotechniques 31:    88-92.-   Yang, H, Leland, J K, Yost, D, Massey, R J (1994):    Electrochemiluminescence: A new diagnostic and research tool.    Biotechnology, 12:193-194.

EXAMPLE 2 Efficacy of an Anti-IL13 Vaccine in a Mouse Asthma Model

The Mouse Asthma Model.

The ovalbumin challenge mouse asthma model is routinely used to assessthe efficacy of asthma therapeutic treatments in vivo. Mice aresensitised with 2 intra-peritoneal doses of ovalbumin given 7 daysapart, which establishes the sensitivity of the mice to ovalbumin. Theasthmatic phenotype can then be generated by giving 3 intra-nasal dosesof ovalbumin. Mice subjected to this protocol exhibit a high level ofairway hyper-responsiveness to the spasmogen 5HT, inflammation of thelung (most notably an eosinophilia of the lung tissue andbroncho-alveolar lavage fluid), and a massive goblet cell metaplasia(and associated mucus hyper-secretion) of the lung airway epithelium.This phenotype mimics that seen in human asthmatics. (Similar mouseasthma models are described in Science 1998 vol 282, pp: 2258-2261 and2261-2263). This model is also described in WO 02/070711.

Anti-IL13 Vaccine Treatment.

Two anti-IL13 vaccine treatments were assessed for efficacy in theovalbumin challenge mouse asthma model, in mice that had previously beensensitised to ovalbumin (Sigma UK Ltd, Poole, Dorset). Both are based onthe mouse chimeric IL13 molecule disclosed in WO 02/070711, which isexpressed and purified as a fusion protein with GST. It is here referredto as gst-cIL13.

1. Vaccine 1=gst-cIL13+‘ImmunEasy’ adjuvant (Qiagen, Cat.No. 303101)

2. Vaccine 2=gst-cIL13+liposomes comprising cholesterol in combinationwith 10 μg. 3-de-O-acylated monophosphoryl lipid A (3D-MPL) and 10 μgQS21 saponin (see EP0822831B1, SmithKline Beecham Biologicals S.A.)

Negative control vaccine treatment groups were also included.

3. Negative control for vaccine 1=gst+‘ImmunEasy’ adjuvant

4. Negative control for vaccine 2=gst+liposomes comprising cholesterolin combination with 10 μg 3-de-O-acylated monophosphoryl lipid A(3D-MPL) and 10 μg QS21 saponin (see EP0822831B1, SmithKline BeechamBiologicals S.A.). Following sensitisation with ovalbumin mice wereimmunised with 4 doses of vaccine, each vaccine dose given 4 weeks apartover a 12 week period. Mice were then challenged with ovalbumin and theasthmatic phenotype assessed.

Other Control Treatment Groups in the Efficacy Study.

A. Dexamethasone (Sigma UK Ltd, Poole, Dorset) is a gold-standardsteroid treatment routinely used in this mouse asthma model. Mice weregiven 3 doses of 1.5 mg/kg dexamethasone via the intra-peritoneal route,during ovalbumin challenge.

B. Passively administered anti-mouse IL13 polyclonal antibody (a proteinA purfied reagent previously made in-house in rabbits) was given as apositive control treatment in this mouse asthma model. A dose ofantibody previously shown to generate fall anti-IL13 driven efficacy inthis mouse asthma model was administered during ovalbumin challenge (=3doses of 0.5 ml of a stock having an endpoint titre of 2×10⁵, forfurther details see WO 02/070711 A1)

C. The maximum phenotype generated by this model was established in anegative control treatment group using saline (Fresenius Kabi,Warrington, UK). Mice were given 3 doses of saline by the intra-nasalroute during ovalbumin challenge. Saline treatment shows no efficacy inthis model, therefore the most severe asthmatic phenotype is generated.

D. As a baseline for comparison of the asthma model phenotype to ‘noinduced asthmatic phenotype’, one treatment group was only sensitisedwith ovalbumin, no ovalbumin challenge doses were given. These miceexhibit normal lung physiology.

Serum IL13 Neutralisation Capacity Generated in Mice Immunised with theAnti-IL13 Vaccines, or Passively Administered Anti-IL13 PolyclonalAntibody.

At the end of the mouse asthma model, mice treated with vaccine orpassively administered anti-IL13 polyclonal antibody, had serum samplesanalysed for IL13 neutralisation capacity using the mouse IL13-inducedTF-1 cell proliferation assay, as described in WO 02/070711. Thisanalysis yields a neutralisation measure termed ND₅₀, which representsthe maximum dilution of mouse serum which is able to reduce by 50% thebioactivity of 5 ng/ml of mouse IL13 in a TF-1 cell proliferation assay.

Our previous data also demonstrated that, using passively administeredneutralising anti-IL13 antibodies, maximal efficacy in this murineasthma model is correlated with a serum ND₅₀ value of approximately1/476. This critical level of neutralisation we term ED₁₀₀ (theeffective neutralising dose required to give 100% efficacy), andcommonly express serum neutralisation capacities relative to this level.For example, a serum sample which had a ND₅₀ of 1/952 would be said tohave a neutralising capacity of 2.0×ED₁₀₀. A sample with a ND₅₀ of 1/238would have a neutralisation capacity of 0.5×ED₁₀₀.

The serum IL13 neutralisation capacity data from this experiment areshown in FIG. 22, and are plotted as a multiples of ED₁₀₀.

All mice that were treated with the chimeric IL13 vaccine or passivelyadminstered with anti-IL13 polyclonal antibody generated serumneutralisation in excess of 1×ED₁₀₀. Therefore it was predicted that themice in these treatment groups would receive full anti-IL13 drivenbenefit in the asthma model.

Airways Hyper-Responsiveness (AHR) Data.

Dose response curves to inhaled spasmogens are used to determine theresponse of the airways to a bronchoconstrictor stimulus. These curvesare comprised of two main components:

1. Hypersensitivity—a leftward shift in the dose response curve (DRC)

2. Hyperreactivity—an increase slope of the DRC and/or a loss in theplateau response

These components together give rise to the general term ‘bronchial orairway hyperresponsiveness’ (BHR or AHR) and this is typically definedas ‘an increase in the ease and degree of airway narrowing in responseto bronchoconstrictor stimuli’.

AHR was measured by challenging conscious mice with a dose of 5HTspasmogen, and then measuring the effects on respiratory flow and volumeparameters using a whole-body plethysmography apparatus (Buxco, Sharon,Conn.). The preferred readout parameter from this analysis is themeasure of enhanced pause (PENH). FIG. 23 illustrates AHR data from thisexperiment obtained by plotting PENH area under curve values for a 5HTspasmogen concentration of 3 mg/ml. Data points are the means andstandard errors for the treatment groups indicated.

Both the vaccine treaments and passively administered anti-IL13polyclonal antibody were as effective as dexamethasone at reducing thelevel of AHR. The negative control vaccine treatments did not reduceAHR.

Lung Inflammation Data.

Lung inflammatory cell content was assessed in the broncho-alveolarlavage fluid (BAL). Average numbers of eosinophils, macrophages,lymphocytes and neutrophils were plotted against treatment received(FIG. 24).

Both the vaccine treaments and passively administered anti-IL13polyclonal antibody were as effective as dexamethasone at reducing thelevel of eosinophils in the BAL fluid. Interestingly, the negativecontrol treatment gst+‘ImmunEasy’ also appeared to effectively reducethe level of BAL eosinophilia. This is probably due to the activity ofthe CpG component in the ‘ImmunEasy’ adjuvant which is known to be animmunomodulatory compound with pro-Th1 activity.

Goblet Cell Metaplasia and Mucus Hyper-Secretion Data.

Mucus containing goblet cells are not normally present at significantfrequencies in the mouse airway epithelium. Following sensitisation andchallenge with ovalubumin in this asthma model, the airway epitheliumbecomes densely packed with mucus containing goblet cells due to ametaplasia of the epithelial layer.

Following fixation, representative samples of the lungs from each animalwere processed for paraffin histology. Sections were cut at 5μ andstained with ABPAS (Alcian blue periodic acid Schiff's reagent,BDH-Merck) with α-amylase (Sigma UK Ltd, Poole, Dorset) pre-digestionfor histopathological evaluation of airway goblet cells (preparativehistology by Propath UK Ltd, Hereford, UK).

The lung sections stained with ABPAS were scored for goblet cell numbersusing the 6-point semi-quantitative scoring system shown below. Theresults are shown in FIG. 25.

Scoring System for Goblet Cells Score Observation 0 No goblet cells 1Very few goblet cells 2 Low numbers of goblet cells 3 Moderate numbersof goblet cells 4 Heavy numbers of goblet cells 5 Massive numbers ofgoblet cells

Note that the scoring system is not linear, and that the differencebetween a score of 2 or 3 is highly significant in relation to thenumber of goblet cells present in the epithelium.

Representative sections for some of the treatment groups are shown inFIG. 26A, gst-cIL13+‘ImmunEasy’; FIG. 26B, gst-‘ImmunEasy’; FIG. 27A,gst-cIL13+Liposomes comprising cholesterol in combination with 10 μg3-de-O-acylated monophosphoryl lipid A (3D-MPL) and 10 μg QS21 saponin(see EP0822831B1, SmithKline Beecham Biologicals S.A.); FIG. 27B,gst+Liposomes comprising cholesterol in combination with 10 μg3-de-O-acylated monophosphoryl lipid A (3D-MPL) and 10 μg QS21 saponin(see EP0822831B1, SmithKline Beecham Biologicals S.A.); FIG. 28,dexamethasone; FIG. 29, maximum asthmatic phenotype.

Both the vaccine treaments and passively administered anti-IL13polyclonal antibody drammatically reduced the numbers ofmucus-containing goblet cells in the airway epithelium. The reduction ingoblet cell number is highly significant for all anti-IL13 treatmentsversus the saline (maximum phenotype) treatment group (<0.01). Negativecontrol vaccines had no effect. Dexamethasone treatment had very littleeffect on goblet cell metaplasia (GCM) in this study.

SUMMARY

The anti-IL13 vaccine treatments were very effective at abrogating theasthmatic phenotype in the mouse asthma model. Anti-IL13 vaccine was aseffective as dexamethasone for treatment of AHR and eosinophilia, andwas superior to dexamethasone for treatment of goblet cell metaplasiaand mucus hyper-secretion.

EXAMPLE 3 Correlation of Goblet Cell Metaplasia with the Level of SerumIL13 Neutralisation Capacity

Some animals immunised with the anti-IL13 vaccines achieved serum IL13neutralisation levels of less than 1.0×ED₁₀₀. To determine whether theseanimals were receiving any discernible benefit (keeping in mind thatED₁₀₀ is defined in terms of maximal benefit), they too were challengedwith ovalbumin, and the degree of GCM determined. The data belowindicates the relationship between goblet cell metaplasia score andlevel of IL13 neutralisation capacity induced in the serum by thevaccine.

Scoring System for Goblet Cells Score Observation 0 No goblet cells 1Very few goblet cells 2 Low numbers of goblet cells 3 Moderate numbersof goblet cells 4 Large numbers of goblet cells 5 Massive numbers ofgoblet cells

The Goblet cell data is shown in table 1 below and in FIG. 30: TABLE 1GCM neut. Mouse score capacity A1 2.5 0.41  2 3 0.3  4 3.5 0.31  8 3.50.21  9 3.5 0 10 2 0.6 11 1.5 0.36 12 3 0.37 14 3 0 15 2.5 0.3 16 2.50.34 18 3 0 20 3.5 0 C30 3 0 31 3 0.21 33 3 0 34 4 0 35 3.5 0 36 3 0 383 0.24 41 2.5 0.36 42 3 0.34 43 3.5 0 45 3 0 46 1.5 0.8 47 2.5 0.31 48 20.26Only mice that generated serum IL13 neutralisation capacity less than1×ED₁₀₀ were included in this analysis, because, by definition, animalswith a serum IL13 capacity equal to or in excess of 1×ED₁₀₀ achieve amaximal efficacy in respect of suppressing goblet cell metaplasia.

The data indicates that there is a correlation between the level ofserum IL13 neutralisation capacity and the severity of goblet cellmetaplasia (R²=0.52). The higher the level of IL13 neutralisation, thelower the severity of goblet cell metaplasia.

These data, together with those of Example 3, validate the use of theED100 measure as a powerful predictor of efficacy of anti-IL13treatments against the asthmatic phenotype. Any vaccine, antibody,soluble receptor or other IL13 neutralising treatment may be evaluatedas follows:

-   1. Administer the IL13 neutralising treatment to the recipient at    the desired dose and frequency.-   2. Take a serum sample.-   3. Determine the IL13 ND₅₀ of the serum sample by analysing it, and    dilutions thereof, in a IL13 bioassay such as the TF1 proliferation    assay. The bioassay is chosen such that it is possible to determine    the greatest serum dilution which causes a 50% inhibition of the    specific effect of 5 ng/ml of mouse IL13. For treatments directed to    human IL13, the TF1 bioassay may still be used, but the stimulating    cytokine will be human IL13 used at a concentration in the range 3-6    ng/ml.-   4. Divide the ND₅₀ value obtained by 1/476 to produce a ED₁₀₀    multiple.-   5. If this multiple is 1.0 or greater, the IL13 neutralising    treatment is expected to have maximal efficacy on the asthmatic    phenotype.-   6. If the multiple is considerably less than 1.0, for example 0.2 or    less, then no significant efficacy is to be expected.-   7. If the multiple lies between these limits, then some efficacy may    be seen, but it will not be optimal, indicating that improvements in    the treatment will be desirable.    This process may be used to guide dose selection for maximal    efficacy. If, after an initial number of doses of agent, the serum    IL13 neutralisation capacity has not reached a level at least equal    to 1.0×ED100, then further doses are given to bring the    neutralisation capacity up to this level.

EXAMPLE 4 Immunogenicity of an Anti-IL13 Protein Vaccine in Combinationwith Various Adjuvants

Studies to investigate the immunogenicity of a gst-cIL-13 immunogen,with or without the additional promiscuous T-cell epitope P30, incombination with several different adjuvants were performed.

gst-cIL13 Protein Immunogenicity Studies

BalbC mice were immunised with 100 μg gst-cIL13 in adjuvant for theprimary immunisation, followed by 50 kg gst-cIL13 in adjuvant for theboost immunisations. Immunisations were administered on a four weeklybasis, serum samples taken from mice 2 weeks after each immunisation (tomonitor the level of IL13 neutralisation capacity generated by theseantibodies in the serum sample). The gst-cIL-13 immunogen was combinedwith four different adjuvants: Group A CpG-2006 adsorbed onto aluminiumhydroxide Group B CpG-1826 Group C CFA prime/IFA boost Group D aluminiumhydroxide

CpG-2006 and CpG-1826 are oligonucelotides containing unmethylated CGdinucleotides, and well-known in the literature for possessingimmunostimulatory activity. CFA/IFA denote complete and incompleteFreunds adjuvant respectively.

The IL13 neutralisation capacity generated by these antibodies in serumsamples was measured in a mouse IL13 bioassay (the TF-1 cellproliferation assay). The table below shows the results (expressed as amultiple of ED₁₀₀) for day 99, post 4 immunisations. The data is alsorepresented graphically in FIG. 31. In this figure, and in the similarfigures that follow, each dot indicates a serum IL13 neutralisationmeasurement for one animal. Animals whose serum neutralising capacity isbelow the sensitivity threshold of the assay (<0.2×ED₁₀₀) are notplotted. IL13 neutralisation capacity expressed as ED₁₀₀ Adjuvanttreatment BalbC mice A B C D 1 <0.2 <0.2 <0.2 <0.2 2 2.7 <0.2 <0.2 <0.23 0.5 <0.2 <0.2 <0.2 4 <0.2 1.4 <0.2 <0.2 5 <0.2 <0.2 <0.2 <0.2

Adjuvant A (CpG (2006) adsorbed onto aluminium hydroxide), incombination with gst-cIL13 protein, was the most effective at generatingneutralising anti-IL13 antibody responses. No neutralising anti-IL13antibody responses were detected for mice treated with gst-cIL13 proteincombined with either alum or CFA/IFA adjuvants.

p30-cIL13 Protein.

Study 1

For this study a different form of IL13 vaccine was used. This isanother chimeric IL13 molecule which contains the p30 epitope fromtetanus toxin at the N terminus. It is encoded by the plasmidpCDNcIL13newFC (FIG. 20), and prepared for vaccine studies as describedin Example 1. The fully processed molecule is termed p30-cIL13 in thedescriptions below.

Five CD-1 mice were immunised with 40 μg p30-cIL13 in adjuvant for theprimary immunisation, followed by 40 μg p30-cIL13 in adjuvant for theboost immunisations. Immunisations were administered on a four weeklybasis, serum samples taken from mice 2 weeks after each immunisation (tomonitor the level of anti-mouse IL13 antibodies present, and the IL13neutralisation capacity generated by these antibodies in the serumsample). As a negative control, serum samples were also analysed fromthree unimmunised CD-1 mice. Group Adjuvant A Immuneasy ™ (purchasedfrom Qiagen Corp.) B liposomes comprising cholesterol in combinationwith 10 μg 3-de-O-acylated monophosphoryl lipid A (3D-MPL) and 10 μgQS21 saponin (see EP0822831B1, SmithKline Beecham Biologicals S.A.). CNo immunisations

Anti-mouse IL13 antibody levels (in a 1/100 dilution of the serumsamples) were measured by ELISA. The table below shows the results(expressed as absorbance at 490 nm) for day 63 post 3 immunisations. Thedata is also represented graphically in FIG. 32, where each barrepresents the data for a single mouse. Absorbance @ 490 nm Mouse ELISAdata 1 2 3 4 5 A 2.654 2.377 2.0995 1.5925 2.4125 B 2.81 2.398 n/a2.6775 2.95 C 0.049 0.0595 0.1095(n/a = sample not available)

Both adjuvants combined with p30-cIL13 protein were able to raiseanti-IL13 antibody responses in CD-1 mice.

The IL13 neutralisation capacity generated by these antibodies in serumsamples was measured in a mouse IL13 bioassay (the TF-1 cellproliferation assay). The table below shows the results (expressed as amultiple of ED₁₀₀) for day 63, post 3 immunisations. The data is alsorepresented graphically in FIG. 33. IL13 neutralisation capacityexpressed as ED₁₀₀ CD-1 mice A B 1 0.755 4.444 2 <0.2 2.963 3 <0.2 n/a 4<0.2 11.429  5 <0.2 3.077

Adjuvant B, in combination with p30-cIL13 protein, was the mosteffective at generating neutralising anti-IL13 antibody responses, 4 outof 5 mice generating potent anti-IL13 neutralising antibody responses inexcess of 1×ED₁₀₀. In comparison, only 1 mouse generated neutralisinganti-IL13 antibody responses when treated with p30-cIL13 proteincombined with ImmunEasy adjuvant (adjuvant A).

Study 2

p30-cIL13 Protein with Oil Emulsion Adjuvant with 3D-MPL and QS21.

Five CD-1 mice were immunised with 40 μg p30-cIL13 in adjuvant for theprimary immunisation, followed by 40 μg p30-cIL13 in adjuvant for theboost immunisations. Immunisations were administered on a four weeklybasis, serum samples taken from mice 2 weeks after each immunisation (tomonitor the level of anti-mouse IL13 antibodies present, and the IL13neutralisation capacity generated by these antibodies in the serumsample). As a negative control, serum samples were also analysed fromthree unimmunised CD-1 mice. Group Adjuvant A ImmunEasy ™ B oil in wateremulsion (oil phase: 1:1 v/v squalene: alpha tocopherol mix,cholesterol + TWEEN 80 ™ surfactant) + 10 μg 3D-MPL and 10 μg QS21) (forfurther details see WO 99/11241 (described as SB62c')) C noimmunisations

Anti-mouse IL13 antibody levels (in a 1/100 dilution of the serumsamples) were measured by ELISA. The table below shows the results(expressed as absorbance at 490 nm) for day 63 post 3 immunisations. Thedata is also represented graphically in FIG. 34. Absorbance @ 490 nmMouse ELISA data 1 2 3 4 5 A 2.654 2.377 2.0995 1.5925 2.4125 B 2.81652.906 2.9035 n/a 3.081 C 0.049 0.0595 0.1095Both adjuvants combined with p30-cIL13 protein were able to raiseanti-IL13 antibody responses in CD-1 mice.

The IL13 neutralisation capacity generated by these antibodies in serumsamples was measured in a mouse IL13 bioassay (the TF-1 cellproliferation assay). The table below shows the results (expressed as amultiple of ED₁₀₀) for day 63, post 3 immunisations. The data is alsorepresented graphically in FIG. 35. IL13 neutralisation capacityexpressed as ED₁₀₀ CD-1 mice A B 1 0.755 3.077 2 <0.2 9.524 3 <0.2 3.3334 <0.2 n/a 5 <0.2 1.176

Adjuvant B, in combination with p30-cIL13 protein, was the mosteffective at generating neutralising anti-IL13 antibody responses, 4 outof 5 mice generating potent anti-IL13 neutralising antibody responses inexcess of 1×ED₁₀₀. In comparison, only 1 mouse generated neutralisinganti-IL13 antibody responses when treated with p30-cIL13 proteincombined with ImmunEasy adjuvant (group A).

Study 3

p30-cIL13 Protein with Oil Emulsion Adjuvant (Without Immunostimulant).

Five CD-1 mice were immunised with 40 μg p30-cIL13 in adjuvant for theprimary immunisation, followed by 40 μg p30-cIL13 in adjuvant for theboost immunisations. Immunisations were administered on a four weeklybasis, serum samples taken from mice 2 weeks after each immunisation (tomonitor the level of anti-mouse IL13 antibodies present, and the IL13neutralisation capacity generated by these antibodies in the serumsample). As a negative control, serum samples were also analysed fromthree unimmunised CD-1 mice. Group Adjuvant A ImmunEasy ™ B oil in wateremulsion (oil phase: 1:1 v/v squalene: alpha tocopherol mix,cholesterol + TWEEN 80 ™ surfactant) (for details see WO9517210) C noimmunisations

Anti-mouse IL13 antibody levels (in a 1/100 dilution of the serumsamples) were measured by ELISA. The table below shows the results(expressed as absorbance at 490 nm) for day 63 post 3 immunisations. Thedata is also represented graphically in FIG. 36, where each barrepresents the data for a single mouse. Absorbance @ 490 nm Mouse ELISAdata 1 2 3 4 5 A 2.654 2.377 2.0995 1.5925 2.4125 B n/a 3.038 1.5625 n/an/a C 0.049 0.0595 0.1095

Both adjuvants combined with p30-cIL13 protein were able to raiseanti-IL13 antibody responses in CD-1 mice.

The IL13 neutralisation capacity generated by these antibodies in serumsamples was measured in a mouse IL13 bioassay (the TF-1 cellproliferation assay). The table below shows the results (expressed as aa multiple of ED₁₀₀) for day 63, post 3 immunisations. The data is alsorepresented graphically in FIG. 37. IL13 neutralisation capacityexpressed as ED₁₀₀ CD-1 mice A B 1 0.755 n/a 2 <0.2 0.32 3 <0.2 0.69 4<0.2 n/a 5 <0.2 n/a

Adjuvant B, in combination with p30-cIL13 protein, was the mosteffective at generating neutralising anti-IL13 antibody responses, 2 outof 5 mice generating anti-IL13 neutralising antibody responses. Incomparison, only 1 mouse generated neutralising anti-IL13 antibodyresponses when treated with p30-cIL13 protein combined with ImmunEasyadjuvant (adjuvant A).

SUMMARY

The ability of the P30 immunogens to augment the immune response in theoutbred CD-1 mouse strain is significant in that is suggests that theuse of this immunogen is not limited to a single immunologicalbackground, and the advantageous effects of P30 should also be obtainedin an outbred human clinical setting.

1. An immunogenic composition comprising an IL-13 element that drives animmune response that recognizes human IL-13 and at least one foreignT-cell epitope.
 2. An immunogenic composition as claimed in claim 1,wherein the T-cell epitope is foreign with respect to humanself-proteins and IL-13 sequence.
 3. An immunogenic composition asclaimed in claim 1, wherein the T-cell epitope is a short peptidesequence added to the IL-13 sequence.
 4. An immunogenic composition asclaimed in claim 3 wherein the carrier protein is selected from thegroup of: Haemophilus influenzae Protein D and CPC (clyta-P2-clyta). 5.(canceled)
 6. An immunogenic composition as claimed in claim 3, whereinat least one short T-cell epitope is added to the IL-13 sequence by anevent selected from the group of: an addition and a substitution.
 7. Animmunogenic composition as claimed in claim 6 wherein the short T-cellepitope is a promiscuous epitope.
 8. An immunogenic composition asclaimed in claim 7 wherein the promiscuous epitope is selected from thegroup of: P2 and P30 from tetanus toxoid.
 9. An immunogenic compositionas claimed in claim 1, wherein the IL-13 element comprises the entirehuman IL-13 sequence.
 10. An immunogenic composition as claimed in claim9 wherein the IL-13 element is in mutated form.
 11. An immunogeniccomposition as claimed in claim 10, wherein the mutated IL-13 is in theform of a chimaeric IL-13 formed by substituting amino acids with aminoacids that are found in equivalent positions within an IL-13 sequencefrom another mammalian species.
 12. An immunogenic composition asclaimed in claim 11, wherein the substitutions occur in areas that areassociated with alpha helical regions.
 13. An immunogenic composition asclaimed in claim 11 wherein the substitutions involve amino acids takenfrom more than one different non-human mammalian species.
 14. Animmunogenic composition as claimed in claim 1 wherein the IL-13 elementis human chimaeric IL-13 sequence having a similar conformational shapeto native human IL-13 and sufficient amino acid sequence diversity toenhance its immunogenicity when administered to a human, wherein thehuman chimaeric IL-13 sequence has the sequence of human IL-13comprising: (a) substitution mutations in at least two of the followingalpha helical regions selected from the group of: PSTALRELIEELVNIT,MYCAALESLI, KTQRMLSGF and AQFVKDLLLHLKKLFRE; (b) comprises in unmutatedform at least six regions of high inter-species conservation selectedfrom the group of: 3PVP, 12ELIEEL, 19NITQ, 28LCN, 32SMVWS, 50SL, 60AI,64TQ, 87DTKIEVA, 99LL, and 106LF; and (c) optionally comprises amutation in any of the remaining amino acids, wherein any substitutionperformed in steps a, b or c is a structurally conservativesubstitution.
 15. An immunogenic composition as claimed in claim 14,wherein greater than 50% of these substitutions or mutations compriseamino acids taken from equivalent positions within the IL-13 sequence ofa non human.
 16. An immunogenic composition as claimed in claim 14,wherein greater than 50% of these substitutions or mutations occur inregions of human IL-13 which are predicted to be alpha helical inconfiguration.
 17. An immunogenic composition as claimed in claim 14,wherein the human chimaeric IL-13 sequence has the sequence of humanIL-13 comprising between 2 and 20 substitutions.
 18. An immunogeniccomposition as claimed in claim 1 wherein the IL-13 element is based ona non-human IL-13 sequence wherein the non-human surface exposed regionsare substituted for the equivalent human sequences.
 19. An immunogeniccomposition as claimed in claim 14, wherein the amino acid sequence ofhuman IL-13 comprises conservative substitutions in at least six of thefollowing positions selected from the group of: 8T, 11R, 18V, 49E, 62K,66M, 69G, 84H, 97K, 101L, 105K, 109E, and 111R.
 20. An immunogeniccomposition as claimed in claim 19 comprising at least six of thefollowing substitutions selected from the group of: 8T to S, 11R to K,18V to A, 49E to D, 62K to R, 66M to I, 69G to A, 84H to R, 97K to T,101L to V, 105K to R, 109E to Q, and 111R to T.
 21. An immunogeniccomposition as claimed in claim 1, wherein the IL-13 element is selectedfrom the group of: Immunogen 1, Immunogen 11, Immunogen 12 and Immunogen13.
 22. An immunogenic composition as claimed in claim 1, selected fromthe group of: Immunogen 2, Immunogen 3, Immunogen 7, Immunogen 8,Immunogen 9 and Immunogen
 10. 23. An immunogenic composition as claimedin claim 1 further comprising a mutation in the human IL-13 element thatabolishes the human IL-13 biological activity and is selected from thegroup of: E12 to I, S, or Y; E12 to K; R65 to D; S68 to D; and R108 toD.
 24. A method of designing an immunogenic composition as claimed inclaim 1 comprising: (a) identifying regions in human IL-13 (SEQ IDNO. 1) that are predicted to form an alpha helical structure; (b)mutating the sequence of human IL-13 within these alpha helical regionsto substitute amino acids from the human sequence with amino acids thatare either a conservative substitution or are found in equivalentpositions within the IL-13 sequence of a different species; and (c)attaching or inserting a source of T-cell epitopes that are foreign withrespect to any human self epitope and also foreign with respect to anymammalian IL-13 sequence.
 25. A method for the manufacture of a humanchimaeric IL-13 immunogen which has a similar conformational shape tonative human IL-13 and sufficient amino acid sequence diversity toenhance its immunogenicity when administered to a human comprising thefollowing steps: (a) performing at least one substitution mutation inhuman IL-13 (SEQ ID NO. 1) in at least two of the following alphahelical regions selected from the group of: PSTALRELIEELVNIT,MYCAALESLI, KTQRMLSGF and AQFVKDLLLHLKKLFRE; (b) preserving at least sixregions of high inter-species conservation selected from the group of:3PVP, 12ELIEEL, 19NITQ, 28LCN, 32SMVWS, 50SL, 60AI, 64TQ, 87DTKIEVA,99LL, and 106LF; (c) optionally mutating any of the remaining aminoacids; and (d) attaching a source of T-cell epitopes that are foreignwith respect to any human self epitope and also foreign with respect toany mammalian IL-13 sequence, wherein any substitution performed insteps a, b or c is a structurally conservative substitution.
 26. Amethod for the manufacture of a human chimaeric IL-13 immunogen asclaimed in claim 25, wherein all four alpha helical regions comprise atleast one substitution mutation.
 27. A method for the manufacture of ahuman chimaeric IL-13 immunogen as claimed in claim 25, wherein thereare no mutations at any region of high inter-species conservation.
 28. Amethod for the manufacture of a human chimaeric IL-13 immunogen whichhas a similar conformational shape to native human IL-13 and sufficientamino acid sequence diversity to enhance its immunogenicity whenadministered to a human, the method comprising the following steps: (a)aligning IL-13 amino acid sequences from different species; (b)identifying regions of high variability and high conservation; (c)mutating human IL-13 (SEQ ID NO. 1) in the areas of high variability tosubstitute amino acids from the human sequence with amino acids that areeither a conservative substitution or are found in equivalent positionswithin the IL-13 sequence of a different species; and (d) attaching asource of T-cell epitopes that are foreign with respect to any humanself epitope and also foreign with respect to any mammalian IL-13sequence.
 29. A method for the manufacture of a human chimaeric IL-13immunogen as claimed in claim 24, wherein all greater than 50% of thesesubstitutions or mutations comprise amino acids taken from equivalentpositions within the IL-13 sequence of a non-human species.
 30. A methodfor the manufacture of a human chimaeric IL-13 immunogen as claimed inclaim 24, wherein greater than 50% of these substitutions or mutationsoccur in regions of human IL-13 which are predicted to be alpha helicalin configuration.
 31. A method for the manufacture of a human chimaericIL-13 immunogen as claimed in claim 24, wherein substitutions ormutations comprise amino acids taken from equivalent positions within atleast two non-human IL-13 sequences.
 32. A method for the manufacture ofa human chimaeric IL-13 immunogen as claimed in claim 24, wherein theimmunogen comprises between 6 and 20 substitutions, and most preferablybetween 6 and 10 substitutions.
 33. An immunogen derived from the methodclaimed in claim 24, wherein the immunogens are immunogenic, whenformulated in an appropriate manner for a vaccine, in a human vaccinee.34. A vaccine comprising the IL13 element as claimed in claim
 1. 35. Apolynucleotide vaccine comprising a polynucleotide that encodes a IL13element as claimed in claim
 1. 36. A method of treating an individualsuffering from or being susceptible to CPD, asthma or atopic dermatitis,comprising administering to said individual a vaccine as claimed inclaim 34, and thereby raising in that individual a serum neutralizinganti-IL-13 immune response and thereby ameliorating or abrogating thesymptoms of COPD, asthma or atopic dermatitis. 37-38. (canceled)
 39. Animmunogenic composition as claimed in claim 1, wherein the T-cellepitope comprises a carrier protein.
 40. An immunogenic composition asclaimed in claim 39, wherein the carrier protein and IL-13 element forma fusion protein.
 41. An immunogenic composition as claimed in claim 3,wherein at least one short T-cell epitope is added to the IL-13 sequenceat a terminal end of the IL-13 sequence by means selected from the groupof: synthetic, recombinant and molecular biology.
 42. An immunogeniccomposition as claimed in claim 1, wherein the IL-13 element comprisesfunctional equivalent fragments of the human IL-13 sequence.